Abstract While potent ART of Simian Immunodeficiency virus (SIV) infected monkeys rapidly contains viral replication to undetectable levels in plasma, our recently developed viral env directed immunoPET/CT imaging technology has been able to detect foyers of continued SIV signals even after prolonged ART. However, the resolution of the technique is restricted to the 1-2 mm range, far from the ?cellular level?, and it remains to be fully adapted and optimized for the detection of HIV. Therefore, here we propose to optimize our immune-PET/CT technology to detect HIV env in vivo both in the context of simian/human immunodeficiency virus (SHIV) infection of macaques under antiretroviral therapy (ART) paving the way for a future translation of the technology to the human clinic. However, to address reservoirs at a cellular and molecular level, we will combine the PET probes with fluorescent moieties emitting in the near-infrared range. Using a Fluobeam, hand-held, near-IR imaging system, we will be able to identify viral reservoirs in organs and tissues and collect the appropriate biopsies and necropsies. Analysis of positive vs negative tissues will follow by confocal, flow cytometry and RNASeq technologies. This will allow us the ability to precisely dissect which cell lineages continue to support residual viral replication under ART, identify their environment, and determine whether the continued replication is secondary to lower local levels of ART, specific activation and/or lack of local antiviral mechanisms. It will also allow for testing whether fully latent viral reservoirs exist in sites that are negative for immunoPET/CT signals. We will first optimize the detection of HIV clades B, C and AE by immunoPE/CT since the optimal probes are anticipated to differ between these clades. We will then focus on the SHIV providing the highest viral load set points to address reservoir longevity under ART in vivo, as well as functional reservoirs from which viral loads will rebound post ART interruption. The use of immunoPET/CT combined with near-IR mapping of viral signal will allow for whole body to single cell analyses of the SHIV reservoir in this model, with comparison of tissues from persistent reservoirs relative to correlated tissues negative for viral signals. This will also permit the delineation of functional differences between these similar tissues, providing mechanistic clues for the longevity of SHIV reservoirs in vivo.